Antibody drugs that are used for the purpose of treating diseases are biodrugs to which advanced bioprocess technologies, including cell line development technology, cell culture technology and purification technology, are applied. As these antibody drugs have been proven to have excellent efficacy and safety compared to existing drugs, the demand therefor has increased day by day. For example, a fusion protein (TNFR:Fc; etanercept) prepared by fusing the extracellular ligand-binding portion of human p75 human tumor necrosis factor receptor to the Fc domain of human IgG1 has been used as an agent for treating rheumatoid arthritis. In addition, in order to increase the in vivo half-life of physiologically active substances, there was an attempt to develop a diabetes therapeutic agent using a fusion protein of a GLP-1R agonist and an Fc domain (US2004-0558627; Chung, H. S. et al., Regulatory peptide, 170, 2011). In addition, there was an attempt to fuse a physiologically active substance such as hGH or EPO to an Fc domain in order to increase the in vivo half-life of the physiologically active substance (KR10-2008-7018012). In recent years, a fusion protein (aflibercept) of a VEGF receptor and an Fc domain has been approved and used as an agent for treating wet age-related macular degeneration (WAMD).
However, the fusion proteins as described above have problems in that they have a large and complex structure and are physically unstable, thus causing aggregation. Various factors that cause such aggregation are present in processes of producing and storing proteins.
For example, aggregation may be caused by one or more of the following factors. During purification, pH, the kind of salt, salt concentration, temperature, contact with air, stirring speed and the like, which are not optimal, may cause aggregation, and during formulation, conditions for concentration of proteins may cause aggregation. During the exchange of buffer, passing through a filter, stirring and the like may cause aggregation, and during storage, a change in temperature, a change in pH, contact with air, stirring and the like may cause aggregation. In addition, when a formulation containing a protein is exposed to light, aggregation may also occur, and the material of a package may also cause aggregation (Hamada, H. et al., Current Pharmaceutical Biotechnology, 10:400, 2009).
The most major aggregation of fusion proteins in buffer occurs when the hydrophobic regions of the antibody protein are exposed due to the structural change thereof. In other words, the hydrophobic regions of protein molecules are gathered together to form an aggregation, and this aggregation may also occur in an irreversible manner due to the formation of covalent bonds between antibody proteins (Hamada, H. et al., Current Pharmaceutical Biotechnology, 10:348, 2009).
Fusion proteins or antibody proteins that aggregated for the above-described reasons generally have reduced activity or lose their activity with the passage of time. In addition, when these proteins aggregate, they have antigenicity that does not appear in a non-aggregated state, and thus can cause the production of antidrug antibody (ADA) when they are injected into the human body. Therefore, there is an urgent need for a method for reducing the aggregation of fusion proteins and a stabilized formulation comprising a fusion protein that exhibits a reduced level of aggregation (Current Trends in Monoclonal Antibody Development and Manufacturing, Biotechnology: Pharmaceutical Aspects Volume XI, 2010, pp 271-291).
For this purpose, a method of either adding an excipient for preventing aggregation or controlling pH or a buffer composition was attempted. In the case in which the hydrophobic groups of an antibody protein are exposed to the surface of the protein molecules and bonded to each other to cause aggregation, an excipient capable of stabilizing the exposed hydrophobic groups is used to prevent the aggregation. For example, there are reports on the use of amino acids such as arginine, lysine, proline, histidine and glycine (U.S. Pat. No. 4,362,661A and U.S. Pat. No. 7,648,702), polysorbate-based surfactants, amphiphilic polymers such as polyethylene glycol (PEG) or polyvinyl pyrrolidone (PVP), polysaccharides such as dextran, or monosaccharides or disaccharides such as sucrose, maltose or trehalose (U.S. Pat. No. 5,945,098A).
However, a fusion protein of a protein and an Fc domain is less stable than generally produced antibody proteins, and thus shows a stronger tendency to form an aggregation compared to other proteins or antibody proteins. There is an example in which a formulation was prepared by adding a surfactant such as polysorbate to the protein-Fc domain fusion protein in order to prevent the aggregation of the fusion protein (Steven J. Shire et al., Biotechnology: Pharmaceutical Aspects, Current Trends in Monoclonal Antibody Development and Manufacturing, XI:115, 2010).
Accordingly, the present inventors have found that a composition containing an ammonium salt or a combination of an ammonium salt and succinate can increase the stability of a fusion protein of a protein and an Fc domain by inhibiting the aggregation of the fusion protein so that it can be easily stored for a long period of time, thereby completing the present invention.